The form of genomic instability associated with defective DNA mismatch repair in tumors is called microsatellite instability (MSI). Microsatellite instability (MSI) is a clonal change in the number of repeated DNA nucleotide units in microsatellites. It typically arises in tumors with defective mismatch repair (MMR) genes: failure of the DNA MMR system to repair errors that occur during the replication of DNA results in accelerated accumulation of single nucleotide mutations and alterations in the length of simple, repetitive microsatellite sequences that occur ubiquitously throughout the genome. MMR-deficiency represents a well-established cause of Lynch syndrome, which is an autosomal dominant inherited disorder of cancer susceptibility that affects 2% tot 5% of endometrial or colorectal cancers. Lynch syndrome is caused by mutations or deletions in the MMR pathway genes (MLH1, MSH2, MSH3, MSH6 or PMS2). Additionally, epigenetic silencing of the MLH1 promoter is responsible for up to 20% of ‘sporadic’ colorectal cancers. Additionally, MMR-deficiency has also been described in a minority of ovarian, pancreatic, gastric, leukemic, as well as several other cancers. Profiling of mutation spectra in cells engineered to have an MMR-deficient (MMR−) system has been widely used to characterize genetic ‘errors’ occurring during DNA replication. Although studies characterizing these mutation spectra have been limited to observations at one or a few reporter loci, or have focused exclusively on mutations at known hotspot sequences, they have helped to understand the mechanisms of the intrinsic DNA repair process. It was found, for instance, that mismatch mutations mostly occur in repetitive DNA sequences, whereby MSH6 is involved in the recognition of insertion-deletions of 1 or 2 bases and MSH3 is responsible for longer insertion-deletion loops.
MMR− tumors are characterized by a distinct response to standard chemotherapies, such as 5-fluoracil and the alkylating agents such as temozolomide. Alternative approaches focusing on the aberrant DNA repair processes of MMR− tumors have therefore been proposed. Synthetic lethality approaches have shown, for instance, that increased oxidative damage (by methotrexate exposure or PINK1 silencing) or interference with the base excision repair (BER) pathway (by DNA polymerase γ or β inhibition) sensitizes MMR− tumors. In particular, in MMR− tumors, oxidative damage induces 8-oxoguanine (8-oxoG) DNA lesions, which fail to be sufficiently repaired either by the BER or MMR pathway, generating mainly GC to TA dinucleotide transversions at the DNA level, leading to cell death. Additionally, it has been hypothesized that there is a maximum mutation frequency that a tumor can tolerate, above which a further increase in mutations would be detrimental. It has therefore been proposed to additionally treat MMR− tumors with mutagenic nucleoside analogues until a critical level of mutations is obtained resulting in error catastrophe-like ablation of the tumor. MMR− tumors are often also resistant to targeted cancer therapies, including anti-EGFR and anti-VEGF therapies. Although the precise reasons for this resistance are unknown, presence of secondary mutations in established tumor driver genes as a consequence of MMR− might be responsible. For instance, MMR− tumors can acquire mutations in double-strand break repair genes (e.g., MRE11, ATR and RAD50), known oncogenes or tumor suppressors (e.g., PIK3CA or PTEN). Since presence of MMR-deficiency mainly in colorectal and endometrial tumors represent a familial form of cancer, and since tumors exhibiting mutation spectra characteristic of MMR-deficiency, diagnostic tests assessing MMR-deficiency are commonly used. By far the most common method to detect MSI is to measure the length of a polymerase chain reaction amplicon containing the entire microsatellite. This requires DNA, a pair of primers of which one is often fluorescently end labeled, a sequencer, and suitable software. Alternatively, if the amplicon is sequenced, one can simply count the number of repeat units. MSI can also be indirectly diagnosed by detecting loss of staining by immunohistochemistry (IHC) of one of the mismatch repair genes, since this also points to an abnormality in mismatch repair. Immunohistochemical and genetic methods are both characterized by a considerable number of false-negatives, and for this reason combined assessments at the immunohistochemical and genetic level are performed in a routine diagnostic setting.
There are at least 500 000 microsatellites in the human genome, and because defective MMR does not affect all microsatellites in a given tumor, it is important to study more than one microsatellite and to study microsatellites that are frequently affected by instability. As microsatellite markers were originally quite randomly picked by researchers, based on their own experiments, a conference was held in Bethesda, Md., to discuss the issues and make suggestions to promote consistency across studies. This resulted in a recommendation for a “golden standard” marker panel, known as the Bethesda panel9. This panel consists of three dinucleotide repeats (D2S123, D5S346, D17S250) and two mononucleotide repeats (BAT26, BAT25) and is still the standard test for MSI. It was proposed to consider a tumor MSI-positive if 40% or more of the markers tested were unstable (also referred to as MSI-high or MSI-H). When using the five-marker panel, this means that MSI is called when at least two of them are positive; however, often four or all five are positive in tumors with MSI. Tumors that test negative for all five markers are termed microsatellite stable (MSS). For tumors that tested positive on 1 tumor marker (or on <30% of tumor markers), the term MSI-L was proposed9.
Although the Bethesda panel is still considered the standard, it is known to have a fairly low sensitivity (also depending on which MMR gene is mutated). For instance, for patients with MLH1 mutations, sensitivity is 80%, but for patients with MSH6 mutations, it is only 55%10. This can be improved by adding further markers10, but still actual MSI-H patients may present as MSI-L or MSS. This is not without significance, as MSI status is important in prognosis (typically better for MSI-H patients11), treatment (MSI-H tumors do not respond to fluoro-uracil(FU)-based adjuvant therapy, as an intact MMR system is needed to induce apoptosis of cells with FU-modified DNA11-13), and diagnosis of several cancers (e.g. those of the Lynch syndrome), and newly diagnosed colorectal cancer (CRC) patients are routinely screened for MSI status.
Another significant disadvantage is that the Bethesda panel is only recommended for colon cancer, even though other cancers displaying MSI are known9. It seems that this is due to the fact that the five markers were rather randomly identified as being mutated in microsatellite unstable colon cancer, but there is no biological mechanism known.
A further disadvantage is of a technical nature. The Bethesda marker panel contains quite long repeats (e.g. the BAT26 marker contains a 26 nucleotide A repeat), and the typical PCR products used to determine MSI status are well over 100 bp. To accurately sequence these fragments and determine the exact length of the repeat, Sanger based sequencing methods in conjunction with multicapillary gel electrophoresis are typically used. However, more and more labs use so called “next generation” sequencing which employ massively parallel sequencing techniques. While cheaper, these technologies make use of shorter reads and cannot be used to detect microsatellite instability on the Bethesda marker panel. As a consequence, labs need to maintain two sequencers: one for Bethesda marker panel screening, and one for other experiments. It would be far more convenient if no special sequencer was required for determining MSI status and this determination could be done on commonly used equipment.
Thus, it would be advantageous to find markers for microsatellite instability that are more sensitive than the currently used Bethesda panel, while retaining specificity for MSI. Ideally, these markers are found using unbiased detection methods (i.e., looking across the whole genome rather than checking specific regions that are supposed to be altered in disease setting). A further advantage would be the identification of markers that are indicative of MSI as such. That is to say, they are a general marker for microsatellite instability, and not just for microsatellite instability in colon cancer (as is the case for the Bethesda panel). This would indeed obviate the need to find new markers for each cancer where MSI can be present. An additional advantage would be the identification of markers whose status can be determined independently of technology. More particularly, markers that can be identified using next generation sequencing technologies (instead of only being identified using Sanger sequencing). This way, labs need not to hold on to an apparatus they only use for checking the Bethesda panel markers.